Stable bortezomib formulations

ABSTRACT

Multi-dose formulations for bortezomib are presented in which bortezomib has significantly improved stability. Especially preferred formulations include those in which bortezomib is in a liquid form suitable for injection, wherein the solvent system predominantly comprises propylene glycol. In other preferred aspects, bortezomib is present as a Lewis donor-acceptor complex with a hetero-bifunctional Lewis base.

This application is a divisional application of our non-provisional U.S.application Ser. No. 13/431,313 (allowed), which was filed Mar. 27,2012, which is a continuation-in-part application of our co-pendingnon-provisional U.S. application Ser. No. 13/051,102, which was filedMar. 18, 2011, and which claims the benefit of priority to U.S.provisional application Ser. No. 61/315,080, which was filed Mar. 18,2010.

FIELD OF THE INVENTION

The field of the invention is bortezomib formulations with improvedstability, and particularly storage-stable multi-dose liquid bortezomibformulations.

BACKGROUND

Bortezomib ((N-(2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronicacid); sold as Velcade™, Millennium Pharmaceuticals) is a 26S proteasomeinhibitor that is approved for use in treating various neoplasticdiseases, and especially treatment of relapsed multiple myeloma andmantle cell lymphoma. It is believed that the boron atom in bortezomibbinds to the catalytic site of the proteasome, ultimately leading toproteasome inhibition and reduced degradation of pro-apoptotic factors,which in turn triggers apoptosis in treated cells. Bortezomib andrelated compounds are described in U.S. Pat. Nos. 5,780,454, 6,083,903,6,297,217, 6,617,317, 6,713,446, 6,747,150, 6,958,319, 7,119,080. Theseand all other extrinsic materials discussed herein are incorporated byreference in their entirety. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Unfortunately, many aminoalkylboronic acids (including bortezomib) willundergo a spontaneous 1,3-rearrangement to give the homologous amines,owing to the instability of free α-amino groups. These compounds yieldboric acids and alcohols by degradation and undergo oxidative reactionsthat easily destroy the C—B bond which is longer and weaker than thecorresponding C—C bond (see e.g., Adele Bolognese, Anna Esposito,Michele Manfra, Lucio Catalano, Fara Petruzziello, Maria CarmenMartorelli, Raffaella Pagliuca, Vittoria Mazzarelli, Maria Ottiero,Melania Scalfaro, and Bruno Rotoli. Advances in Hematology, 2009 (2009)1-5). Such instability is borne out in stress testing and acceleratedstability studies of bortezomib that has established that bortezomib inaqueous solution for injection is intrinsically unstable. For example,in an ethanol:normal saline solution (2:98, pH 2.8), Bortezomib (0.5mg/mL) degraded 20% at 25° C. in 1 month, and in propyleneglycol:ethanol:water (50:10:40), the stability of the compound improved,but still degraded 20% in 8 months when stored at 25° C. Among otherfactors, it was speculated that the degradation of Bortezomib observedin PEG300:EtOH:H2O (40:10:50) solvent might be due to the presence ofperoxides, as PEG300 is known to undergo auto-oxidation with concomitantperoxide generation. (Journal of Pharmaceutical Sciences, 89, 2000758-765).

In other studies, bortezomib was reported to be susceptible to oxidativedegradation under a number of experimental conditions, and that theoxidation of alkyl boranes (which yields the ester of boric acid) canalso be due to reaction with alkyl peracids, alkyl peroxides, or oxygenradical species. (Brown H C. 1972. Boranes in organic chemistry. Ithaca,N.Y.: Cornell University Press.) The initial oxidation can be attributedto peroxides or molecular oxygen and its radicals and as light, metalions, and alkaline conditions normally facilitate oxidation. Theseconditions are therefore not considered favorable to the stability ofbortezomib or any other alkyl boronic acid derivative. (Hussain M A,Knabb R, Aungust B J, Kettner C. 1991. Anticoagulant activity of apeptide boronic acid thrombin inhibitor by various routes ofadministration in rats. Peptides 12:1153-1154).

Formation of boronic esters from diol and polyols was reported byKuivila et al. reporting the preparation of several esters ofphenylboronic acid by reaction with sugars like mannitol and sorbitol,and 1,2-diols like catechol and pinacal. (J. Org. Chem. 1954, 8,780-783), and reversible formation of boronic ester by the interactionof boronic acids and polyols in water was first noted by Lorand andEdwards. (J. Org. Chem. 1959, 24, 769-774). U.S. Pat. Nos. 7,119,080,6,713,446, 6,958,319, 6,747,150, and 6,297,217 disclose formation ofdiester of boronic acid functional group with mannitol afterlyophilization. From the so formed ester, the active boronic acid isobtained upon reconstitution of the drug product in saline solution forinjection. Similarly, attempts to form the ester of boronic acid withalpha-hydroxy and beta-carboxylic acids like citric acid along withbulking agents and buffers was disclosed in WO 2009/154737.

To circumvent issues with stability of bortezomib in solution, thecompound can be lyophilized and reconstituted prior to injection.However, while such an approach tends to solve the issues associatedwith bortezomib stability, unused reconstituted solution must beinjected within hours or days (see e.g., Stability of unusedreconstituted bortezomib in original manufacturer vials; J Oncol PharmPract. 2010 Oct. 6, or Stability of bortezomib 1-mg/mL solution inplastic syringe and glass vial; Ann Pharmacother. 2005 September;39(9):1462-6). Similarly, bortezomib esters of mannitol whenreconstituted are suitable only for administration within 8 hr whenstored at room temperature. Still further known approaches includeisolation of specific polymorphic forms having improved stability asdescribed in WO2008075376A1, and lyophilized forms with tromethamine asdescribed in WO2010089768A2. Yet other formulations with selectedorganic solvents and other ingredients are described in WO2010039762A2.Unfortunately, all or almost all of such known compositions fail toprovide significant stability for bortezomib, especially storagestability where the formulation is a liquid formulation.

Therefore, even though there are many formulations for bortezomib knownin the art, all or almost all of them suffer from limited stability whenbortezomib is in solution, particularly over extended periods.Consequently, currently used products fail to provide flexibility ofdosing. More importantly, the currently known or marketed products donot allow for ready-to-use multi-dose liquid formulations havingextended stability. Thus, there is still a need to provide improvedliquid bortezomib formulations with greater stability.

SUMMARY OF THE INVENTION

The inventive subject matter is drawn to compositions and methods forbortezomib in solution in which bortezomib has significantly increasedstability over prolonged periods of time. In most preferred aspects,contemplated formulations are single phase, substantially non-aqueousliquid formulations, and/or formulations in which bortezomib isformulated with a hetero-bifunctional Lewis base donor compound to forma Lewis donor-acceptor complex.

In one preferred aspect of the inventive subject matter, a liquid singlephase multi-dosage pharmaceutical composition is formulated and providedin a suitable container for single or multiple use that includes aliquid formulation comprising bortezomib, wherein the liquid formulationis a substantially non-aqueous solvent system suitable for injection,and wherein the solvent system comprises as a main component propyleneglycol. As used herein, the term “multi-dosage” and “multi-dose” areused interchangeably herein, and when used in conjunction with apharmaceutical composition or formulation, refer to a pharmaceuticalcomposition or formulation that has a volume and/or quantity of theactive pharmaceutical ingredient suitable for at least two independentand distinct administrations (to the same or a different patient) of thepharmaceutical composition or formulation. Most preferably, thebortezomib in such formulations is present at a pharmaceuticallyeffective concentration and in an amount sufficient for at least twoindependent dosages, and the solvent system is formulated to maintaindegradation of the bortezomib at a level of less than 10 wt % (moretypically equal or less than 8 wt %, and most typically 2-6 wt % andeven lower) when the liquid formulation is stored over at least threemonths at ambient conditions (i.e., 25° C., 60% relative humidity).

It is especially preferred that the substantially non-aqueous solventsystem comprises at least 50 vol %, more preferably at least 75 vol %,and most preferably 100 vol % propylene glycol. In such formulations, itis still further preferred that the substantially non-aqueous solventsystem further comprises a polar solvent in an amount of equal or lessthan 50 vol %, more preferably equal or less than 25 vol %, and mostpreferably equal or less than 15 vol %. Among other choices, the polarsolvent is most preferably ethanol. Alternatively, the substantiallynon-aqueous solvent system may include the polar solvent in an amount ofequal or less than 15 vol %, and more typically equal or less than 10vol %. In such case, the polar solvent is preferably water.

In another preferred aspect of the inventive subject matter, apharmaceutical composition comprises bortezomib and ahetero-bifunctional Lewis base, wherein the bortezomib and thehetero-bifunctional Lewis base together are present in form of a Lewisdonor-acceptor complex, and wherein especially preferredhetero-bifunctional Lewis bases have at least two distinct donor groups(most preferably selected from —NH₂, —SH, COOH, and —OH). Suchcontemplated formulations will preferably be lyophilized or in solution.

It is generally preferred that in such formulations bortezomib and thehetero-bifunctional Lewis base are present in a ratio of 1:200, morepreferably in a ratio of 5:80, and most preferably in a ratio of 20:40.Most typically, preferred hetero-bifunctional Lewis bases include aminoacids (e.g., naturally occurring amino acid or an N-acetylated aminoacid), peptides (e.g., naturally or synthetic dipeptides ortripeptides), and substituted polyethylene glycols. Particularlypreferred substituted polyethylene glycol have a structure according toFormula I

wherein n is an integer between 2 and 5,000, and wherein each A isindependently selected from the group consisting of hydrogen, —NH2, —SH,—COOH, and —OH. Where the composition is lyophilized, it is preferredthat the formulation includes a buffering agent, a lyoprotectant, acryoprotectant, a preservative, and/or an antioxidant.

In yet another aspect of the inventive subject matter, a storage-stableliquid pharmaceutical composition includes bortezomib in a single-phaseliquid formulation comprising a substantially non-aqueous solvent systemsuitable for injection, a buffer, and bortezomib, wherein the bortezomibis present in the formulation at a therapeutically effectiveconcentration (e.g., between 1 mg/ml and 5 mg/ml). In especiallypreferred compositions, the solvent system comprises as a predominantcomponent (i.e., at least at 50 vol %, more typically at least 70 vol %,most typically at least 90 vol %) propylene glycol. Moreover, it iscontemplated that the solvent system, the buffer, and the pH areselected such as to be effective to suppress formation of at least oneof an amide degradation product, a first carbinolamide degradationproduct, and a second carbinolamide degradation product when the liquidformulation is stored under storage conditions (e.g., at least 15 daysat 50° C.). Viewed from a different perspective, one particularlypreferred non-aqueous solvent system consist essentially of propyleneglycol, or comprises at least 70 vol % (and more typically at least 90vol %) propylene glycol, and includes as especially preferred buffer anaqueous acetate buffer (e.g., at a concentration of between 0.05 and0.25M), particularly at pH 3.

Thus, the inventors also contemplate a container (e.g., a vial, anampoule, an intravenous bag, or a syringe) that may or may not beconfigured as a multi-use container. In such uses, the containerincludes a quantity of the liquid formulation that is suitable forindependent and multiple administrations.

Viewed from another perspective, the inventors also contemplate a methodof suppressing formation of a plurality of degradation products ofbortezomib in solution. Especially preferred methods include a step ofcompounding a single-phase liquid formulation from a substantiallynon-aqueous solvent system suitable for injection, a buffer, andbortezomib, wherein the bortezomib is present in the formulation at apharmaceutically effective concentration (preferably between 1 mg/ml and5 mg/ml). Most preferably, the solvent system essentially consists of orcomprises as a main component propylene glycol, and the solvent system,the buffer, and the pH are selected such as to be effective to suppressformation of at least one of an amide degradation product, a firstcarbinolamide degradation product, and a second carbinolamidedegradation product when the liquid formulation is stored under storageconditions (e.g., storage at 50° C. over 15 days). In especiallypreferred methods, the buffer is an aqueous acetate buffer at aconcentration of between 0.05 and 0.25M and the pH of the formulation ispH 3.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION

The present invention is generally directed towards to pharmaceuticalcompositions and methods of preparing liquid and lyophilizedformulations containing therapeutically effective concentrations ofbortezomib, where the formulation provides significantly improvedstability for bortezomib, especially where the formulation is a liquidformulation. Where the formulation is lyophilized or concentrated abovethe concentration suitable for injection, contemplated compositions willbe administered after reconstitution with one or more pharmaceuticallyacceptable diluents, optionally further containing pharmaceuticallyacceptable antioxidants, stabilizers, preservatives and/or co-solvents.

In certain aspects of the inventive subject matter, contemplatedformulations will include bortezomib and a hetero-bifunctional Lewisbase donor to so form a donor acceptor complex, while in other aspectscontemplated formulations are liquid formulations and will include an atleast binary non-aqueous solvent system. In still further contemplatedaspects, bortezomib and/or bortezomib donor acceptor complexes may alsobe encapsulated in a pharmaceutically acceptable delivery or carriersystem, particularly in liposomes, micelles, nanoparticles,microspheres, emulsions, and/or suspensions. Regardless of theparticular form of preparation, contemplated formulations may furtherinclude stabilizing agents, buffer components, antioxidants, isotonicityadjusting agents and lyoprotective agents.

Most typically, contemplated pharmaceutical formulations are stable formonths at ambient conditions (i.e., 25° C., 60% relative humidity) whenstored in an amber vial with nitrogen head space. Most typically,contemplated formulations will be subjected to sterile filtration, andwhen lyophilized, can be reconstituted with intravenous diluents such assaline, dextrose, or water for injection.

For example, in one preferred aspect, contemplated pharmaceuticalcompositions will include a liquid formulation that includes bortezomibin a substantially non-aqueous solvent system suitable for injection,and wherein the solvent system comprises propylene glycol as a maincomponent. The term “substantially non-aqueous solvent system” refers toa solvent system in which bortezomib is completely soluble without waterat a concentration of up to 10 mg/ml and that comprises water in a totalamount of equal or less than 15 vol %. Where desired an antioxidant maybe included in the formulation. As also used herein, the term “singlephase” in conjunction with a solvent system refers to a composition inwhich multiple components do not separate into or exist in distinctphases. Therefore, liposomal formulations, emulsions, and suspensionsare not considered single phase solvent systems. On the other hand, amixture of two or more solvents that are miscible with each other areconsidered a single phase solvent system. In another preferred example,contemplated pharmaceutical compositions will include a formulation inwhich bortezomib and a hetero-bifunctional Lewis base form a Lewisdonor-acceptor complex. Most typically, the hetero-bifunctional Lewisbase has at least two distinct donor groups (preferably selected fromthe group of —NH₂, —SH, COOH, and —OH), and the formulation islyophilized or in solution. As used herein, the term “donor acceptorcomplex” refers to a non-covalent and non-ionic association with astability that is intermediate with respect to stability of covalent andionic bonds.

Most preferably, bortezomib and the hetero-bifunctional Lewis base arepresent in a ratio of 1:100 to 1:200, more typically 1:10 to 1:100, andmost typically 1:1 to 1:10. Unless the context dictates the contrary,all ranges set forth herein should be interpreted as being inclusive oftheir endpoints, and open-ended ranges should be interpreted to includecommercially practical values. Similarly, all lists of values should beconsidered as inclusive of intermediate values unless the contextindicates the contrary.

In another preferred aspect, a storage-stable liquid pharmaceuticalcomposition is contemplated that includes bortezomib in atherapeutically effective amount. As used herein, the term“storage-stable liquid pharmaceutical composition” refers to a liquidpharmaceutical composition in which the pharmaceutically activeingredient (bortezomib) is dissolved in a solvent or solvent system(which may comprise a buffer) at a ready-to-use concentration, and inwhich at least 99% of the pharmaceutically active ingredient remain inan undegraded state after storage of the composition over seven days at50° C.

Most preferably, the composition comprises a single-phase liquidformulation comprising a substantially non-aqueous solvent systemsuitable for injection, a buffer, and bortezomib, wherein the bortezomibis present in the formulation at a therapeutically effectiveconcentration. In most preferred aspects of the inventive subjectmatter, bortezomib will therefor be present at a concentration ofbetween 0.1 mg/ml to 10.0 mg/ml, and more typically between 0.5 mg/mland 5.0 mg/ml, and most typically between 1.0 mg/ml and 2.5 mg/ml,inclusive.

In particularly preferred aspects, the solvent system in suchformulations has as a single and predominant component propylene glycol.Thus, the solvent system in especially preferred formulationsessentially consists of propylene glycol. However, in less preferredaspects, the formulation may also include one or more additionalsolvents that are miscible with propylene glycol, and especiallypreferred co-solvents include polyethylene glycol and ethanol.Therefore, contemplated formulations will comprise at least 70 vol %,and more typically at least 90 vol % propylene glycol. As can be seenfrom the experimental data below, the chemical stability of bortezomibcan be greatly increased by appropriate choice of the solvent system.

Likewise, while numerous pharmaceutically acceptable buffers are deemedappropriate for use herein, especially preferred buffers are aqueousbuffers, and especially acetate buffer (see also results below). Withrespect to the strength of the buffer, it is generally preferred thatthe buffer is present in a concentration of between 0.01M and 0.5M, moretypically between 0.025M and 0.3M, and most typically between 0.05M and0.2M. The buffer will most preferably have a pH of 3.0, however,moderate modifications to that pH value are also contemplated. As isshown in more detail below, the inventors have discovered thatcontemplated bortezomib formulations have significantly and unexpectedlyhigh stability at a pH that is about 3.0 in aqueous acetate buffer,particularly where the formulation has a substantially non-aqueoussolvent system that has as a single and predominant component propyleneglycol. Of course, it should be appreciated that similarly highstability may also be achieved with small deviations around pH 3.0.Determination of the preferred stability range around pH3 using acetatebuffer can be performed without undue experimentation. Therefore,suitable pH values of the acetate buffer will typically also include pHranges between 2.7 to 3.0 and 3.0 to 3.3.

Based on the experimental data below, the inventors thereforecontemplate formulations that include bortezomib, a solvent system, anda buffer at a pH, wherein the solvent system and the pH are selectedsuch as to be effective to suppress formation of an amide degradationproduct, a first carbinolamide degradation product, and/or a secondcarbinolamide degradation product when the liquid formulation is storedunder storage conditions. As used herein, the term “suppress formation”with respect to degradation products (amide degradation product, firstcarbinolamide degradation product, and/or second carbinolamidedegradation product) means that there is no detectable quantity (using amodified HPLC assay method as published in Journal of PharmaceuticalSciences, 89, 2000, 758-765) of at least one of the degradation productsin a formulation after storage over a period of at least seven days at atemperature of 50° C. Chromatographic conditions for the HPLC analysisof Bortezomib formulations used a Symmetry column, Waters C-8, 3.5μ,4.6×150 mm at a column temperature of 30° C. Mobile phase was68/32:water/acetonitrile containing 0.1% formic acid and 0.05%triethylamine at a flow rate of 1.0 mL/min and isocratic elution. UVDetection was performed at 270 nm, and injection volume was 10 μL.

It should be particularly noted that storage stability of liquidbortezomib composition is particularly important as bortezomib is highlyinstable in a wide variety of liquid solvents and conditions as isreadily evident from published data and the experimental data below. Asa consequence, most commercially available bortezomib compositions aretherefore lyophilized compositions that need reconstitution with asolvent. However, once reconstituted, such solutions cannot be storedover extended periods without significant degradation, which precludesmulti-dose liquid formulations for use in multiple patients and/or usefor a single patient over a prolonged treatment period. Degradation ofbortezomib in solution is a well-known phenomenon and an exemplarydegradation scheme is depicted in Scheme I below. Here, compound II is afirst carbinolamide degradation product, compound III is a secondcarbinolamide degradation product (which is a stereoisomer of II).Hydrolysis of II or III will lead to the formation of the correspondingamide IV, which can be further hydrolyzed to the carboxylic acid productV.

Therefore, and viewed from another perspective, the present inventivesubject matter is drawn to compositions and pharmaceutical formulationscomprising bortezomib in a stable liquid dosage form or as a stablelyophilized product. In most instances, the inventors contemplate thatthe pharmaceutical formulations in liquid forms provide stability ofbortezomib at ambient conditions for at least two, more typically six,even more typically 12, and most typically 24 months and even longer. Asfurther shown below (see examples, further data not shown), contemplatedformulations provided significant stability to bortezomib in varioussolvent systems, and preferred solvent systems were formulated such thatdegradation of bortezomib was maintained at or below 10 wt %, moretypically at or below 8 wt %, even more typically at or below 6 wt %,and most typically at or below 4 wt % and even at or below 2 wt % wherethe liquid formulation was stored over at least three months at ambientconditions.

With respect to storage stability, the inventors used model conditionswell known in the art to predict or extrapolate storage stability underambient conditions. For example, as can be seen from the experimentaldata below, the inventors used ‘accelerated’ storage conditions whereformulations were stored at 40° C. and 75% relative humidity, and ‘superaccelerated’ storage conditions where formulations were stored at 50° C.and 75% relative humidity for one month, which typically allows topredict or extrapolate corresponding stability data where theformulations is stored at ambient conditions for 16 months.

Similarly, where bortezomib is in lyophilized form, contemplated formswill provide stability of bortezomib at ambient conditions for at leasttwo, more typically 6, and most typically 12 months and even longer. Itshould be appreciated that bortezomib may be present in contemplatedpharmaceutical formulations in any suitable amount, and most preferablyin an amount that is suitable for injection after reconstitution. Thus,and viewed from a different perspective, bortezomib is present in atherapeutically effective amount to treat a neoplastic (or other)condition in a human or other non-human mammal. In preferred aspects,bortezomib is present in a therapeutically effective amount to treatcancer. Typically, the bortezomib is present in an amount of about 0.01%to about 99% w/w of the total composition.

In especially preferred aspects, the non-aqueous solvent system is asingle solvent or a binary solvent system, which may optionally furtherinclude a buffer. While various alternative solvents are also deemedsuitable for use herein, particularly preferred solvents and solventsystems include propylene glycol, one or more short chain alcohols(C₁-C₆), dimethyl acetamide, N-methyl pyrrolidone, dimethyl sulphoxide,and glycerol. Viewed from a different perspective, suitable solventsespecially include polar non-protic and protic solvents. Where thesolvent system is a binary system it is preferred that the solvents aretwo or more of short chain alcohols (e.g., ethanol, tert-butyl alcohol),aryl alcohols (e.g., benzyl alcohol), glycols (and especially propyleneglycol), dimethyl acetamide N-methyl pyrrolidone, and dimethylsulphoxide.

Unexpectedly, the inventors further discovered that certain solventsallowed formation of a stable and liquid formulation, while closelyrelated solvents lead to rapid degradation. For example, and as can beseen further below, propylene glycol allowed for formation of a stablesolution of bortezomib while solutions with polyethylene glycol oftenlead to rapid degradation of the bortezomib. Similarly, ethanol inrelatively low concentrations (e.g., equal or less than 25 vol %, moretypically equal or less than 20 vol %) afforded a more stableformulation while ethanol quantities above 25 vol % led to markeddegradation. It should further be appreciated that especially preferredsolvents (e.g., propylene glycol, ethanol) will not lead to ester ordi-ester formation, even in formulations with low (e.g., equal or lessthan 15 vol %) or no water content.

Likewise, it should be noted that bortezomib will not form an ester ordi-ester with a (hetero-)bifunctional Lewis base donor molecule.Instead, bortezomib will form in most cases a donor acceptor complexthat is intermediate in stability to an ionic bond and a covalent bond.Thus, the boronic acid moiety remains protected in solution or inlyophilized state without ester formation, leading to significantlyimproved stability. For example, suitable hetero-bifunctional Lewis basedonors include compounds with two or more —OH, —SH, —COOH, and/or —NH2groups, which are most typically vicinal groups or separated by no morethan 4 atoms in linear dimension. For example, suitablehetero-bifunctional Lewis base donors include compounds includecompounds in which the two hetero-functional groups are —OH and —SH, —OHand —NH2, —SH and —NH2, —COOH and —NH2, and —COOH and —SH.

A large variety of hetero-bifunctional Lewis base donors are known inthe art and especially preferred donors include numerous amino acids(e.g., proteinogenic, essential, non-essential, chemically modified,synthetic, beta-, gamma-amino acids, etc. acids), all of which may be inD- or L-configuration. For example, contemplated amino acids includealanine, asparagine, aspartic acid, arginine, cysteine, glutamine,glycine, glutamic acid, histidine, isoleucine, lysine, leucine,phenylalanine, methionine, serine, proline, tryptophan, threonine,tyrosine and valine.

In further examples, the hetero-bifunctional Lewis base donor may alsobe a synthetic or natural peptide, and especially a dipeptide, atripeptide, or an oligopeptide. Examples of peptides include carnosine,anserine, homoanserine, kyotorphin, balenine, aspartame, glorin,barettin, pseudoproline, glycylglycine, isoleucine-proline-proline(ipp), glutathione, thyrotropin-releasing hormone, melanostatin,ophthalmic acid, leupeptin, and eisenin. Oligopeptides are also deemedsuitable, albeit less preferred.

In still further contemplated examples, hetero-bifunctional Lewis basedonors may also be various polymers with pendant and/or terminal Lewisbase donor groups. Among other preferred choices, especially suitablepolymers include pharmaceutically acceptable polymers, includingsubstituted polyethylene glycols with structure according to Formula I

wherein n is an integer between 2 and 5,000, and wherein each A isindependently selected from the group consisting of hydrogen, —NH₂, —SH,COOH, and —OH. In still further preferred aspects, the polymer may alsocomprise a carbohydrate backbone that is derivatized with two or moredistinct Lewis donor groups. Of course, it should be appreciated thatall polymers are especially that are pharmaceutically acceptable.

It should be further noted that the complex of the hetero-bifunctionalLewis base donor with bortezomib may be formed in numerous manners, andparticularly suitable manners include heating in a solvent of choice foran appropriate period of time. Alternatively, complexes or esters canalso be prepared by evaporation of solvent, salting out, orprecipitation (facilitated by seeding). A further especially preferredmanner is co-lyophilization of bortezomib with a hetero-bifunctionalLewis base donor, typically from an aqueous solution comprising ofbortezomib and a molar excess of the hetero-bifunctional Lewis basedonor. In some embodiments, the aqueous solution additionally comprisesa (preferably water-miscible) co-solvent. Example of suitableco-solvents includes, but not limited to tert-butyl alcohol, methanol,ethanol, and mixtures thereof. While the molar excess of thehetero-bifunctional Lewis base donor relative to bortezomib can be in awide range, it is generally preferred that the excess is between 1:1 to1:200, more typically 1:100 to 1:200, even more typically 1:10 to 1:100,and most typically 1:1 to 1:10.

Depending on the particular formulation, contemplated compositions maycomprise one or more bulking agent, cryoprotectant, or lyoprotectants tofacilitate lyophilization. In some embodiments, the Lewis base donormolecule may also act as a bulking agent, cryoprotectant,lyoprotectants, and/or stabilizer. Further suitable lyoprotectantsincluding amino acids, and polymers. Preferably, amino acids will beselected from lysine, alanine, glycine. Suitable polymers includevarious proteins (e.g., gelatin, albumin, etc.), polyethylene glycol,polyvinyl pyrrolidone, and Dextran-40. Most typically, the lyoprotectantrepresents less than 50% w/w of the total composition, and allconcentrations above 1% w/w of the total composition are deemedeffective to enhance the stability of the formulation. Thus, thelyoprotectant may be present in an amount of at least about 5% w/w, atleast about 10% w/w, or at least about 20% w/w of the total composition.

The compositions contemplated herein may further include tonicityagents, and suitable tonicity agents include sodium chloride, glycerol,thioglycerol. Additionally, contemplated formulations may includefurther pharmaceutically acceptable excipients, and especially buffers,preservatives, and antioxidants, and any reasonable mixture thereof.However, in at least some formulations, the inventors unexpectedlydiscovered that formulations without antioxidants (and particularlywithout N-acetyl cysteine) had increased stability.

It should further be appreciated that depending on the particularingredients, the pH of the formulation may vary. However, it isgenerally preferred that the pH of the formulations is suitable forinjection and will typically be between 4.0 and 9.0, more typicallybetween 5.5 and 8.0. Thus, one or more buffer systems may be employed tostabilize the pH at a desired value or range. Suitable buffers includecitric acid buffer, acetic acid buffer, maleic acid buffer, phosphoricacid buffer, succinic acid buffer, and tartaric acid buffer. Mosttypically, the buffer strength is between 5 mM to 150 mM, however,higher and lower strengths are also deemed suitable for use herein.Remarkably, significant increases in stability were also observed wherethe solution was buffered with acetate buffer at pH 3 (or close to pH 3,typically between pH 2.7-3.3).

To still further improve the stability, the formulations may alsoinclude one or more antioxidants. For example, hydrophobic anti-oxidantsinclude butylated hydroxytoluene, butylated hydroxyanisole, propylgallate, and α-tocopherol, DL-tocopherol, α-tocopherol acetate,Tocopherol Polyethylene Glycol Succinate (Vitamin E TPGS), L-cysteine,or hydrophilic anti-oxidants, including sodium EDTA and thioglycerol.Most typically, the concentration of the antioxidant will be between0.005% and 5% w/w of the total composition. Additionally, oralternatively, contemplated formulations may include a preservative(e.g., phenol, thimerosal, chlorobutanol, benzyl alcohol, m-cresol,phenoxyethanol, methylparaben and propylparaben), typically at aconcentration of between 0.001% w/w and less than 5% w/w of the totalcomposition, and most typically between 0.003% and 2.0% w/w of the totalcomposition.

It should further be appreciated that contemplated formulations will besterilized and all known manners of sterilization are deemed suitablefor use herein, including filtration through 0.22 micron filters, heatsterilization, radiation (e.g., gamma, electron beam, microwave), and/orethylene oxide sterilization to render the formulations sterile. Wherecontemplated formulations are lyophilized, they may be prepared aslyophilized cake, lyophilized powder, etc. The solutions or lyophilizedforms may be diluted and/or reconstituted with standard intravenousdiluents known in the art. For example, suitable intravenous diluentsfor use in the present invention include water, saline, dextrose 5% inwater, water for injection or lactated ringer's solution.

Therefore, the inventors particularly contemplate a liquidpharmaceutical composition that includes bortezomib in a therapeuticallyeffective amount, a substantially non-aqueous solvent system suitablefor injection, and an aqueous buffer, wherein the composition has a pHthat is equal or less than 4.0. In one especially preferred aspect, theliquid pharmaceutical composition comprises (a) bortezomib at aconcentration of between 0.1 mg/ml to 10 mg/ml, more typically between0.5 mg/ml to 5.0 mg/ml, and most typically between 1 mg/ml to 2.5 mg/ml,(b) propylene glycol as a predominant component (typically at least 50vol %, more typically at least 70 vol %, even more typically at least 90vol %, and most typically essentially consisting of propylene glycol) ofthe substantially non-aqueous solvent system, and (c) a non-chelatingbuffer or mono-dentate buffer, most preferably acetate buffer at abuffer strength of between 0.01 and 0.5M, and more typically between0.05 and 0.25M, wherein the buffer has a pH of between 2.0 and 4.0, moretypically between 2.3 and 3.7, and most typically between 2.7 and 3.3.Such compositions will exhibit equal or less than 10% degradation ofbortezomib at accelerated storage conditions after 3 months, and morepreferably equal or less than 7% degradation of bortezomib ataccelerated storage conditions after 3 months, even more preferablyequal or less than 5% degradation of bortezomib at accelerated storageconditions after 3 months, and most preferably equal or less than 3%degradation of bortezomib at accelerated storage conditions after 3months. Thus, and viewed from a different perspective, contemplatedcompositions are storage-stable liquid pharmaceutical compositions.

For example, particularly preferred liquid pharmaceutical compositionscomprise bortezomib at a concentration of between 1 mg/ml to 2.5 mg/ml,include propylene glycol in an amount of at least 70 vol % of thesubstantially non-aqueous solvent system, and further include acetatebuffer at a buffer strength of between 0.05 and 0.25M, wherein thebuffer has a pH between 2.7 and 3.3. Such compositions will exhibitequal or less than 10% degradation of bortezomib at accelerated storageconditions after 3 months.

In another example, particularly preferred liquid pharmaceuticalcompositions comprise bortezomib at a concentration of between 0.5 mg/mlto 5.0 mg/ml, include propylene glycol in an amount of at least 90 vol %of the substantially non-aqueous solvent system, and further includeacetate buffer at a buffer strength of between 0.05 and 0.25M, whereinthe buffer has a pH between 2.7 and 3.3. Such compositions will exhibitequal or less than 10% degradation of bortezomib at accelerated storageconditions after 3 months. In yet another example, particularlypreferred liquid pharmaceutical compositions comprise bortezomib at aconcentration of between 1 mg/ml to 2.5 mg/ml, include propylene glycolin an amount of at least 70 vol % of the substantially non-aqueoussolvent system, and further include a non-chelating buffer (e.g.,monocarboxylic acid buffer) or mono-dentate (i.e., only one complex bondis formed between one chemical group of the buffer molecule and theboron moiety of bortezomib) buffer at a buffer strength of between 0.05and 0.25M, wherein the buffer has a pH between 2.3 and 3.7. Suchcompositions will exhibit equal or less than 10% degradation ofbortezomib at accelerated storage conditions after 3 months.

In still another example, particularly preferred liquid pharmaceuticalcompositions comprise bortezomib at a concentration of between 1 mg/mlto 2.5 mg/ml, include propylene glycol in an amount of at least 90 vol %of the substantially non-aqueous solvent system, and further includeacetate buffer at a buffer strength of between 0.05 and 0.25M, whereinthe buffer has a pH between 2.3 and 3.7. Such compositions will exhibitequal or less than 10% degradation of bortezomib at accelerated storageconditions after 3 months.

In yet a further example, particularly preferred liquid pharmaceuticalcompositions comprise bortezomib at a concentration of between 1 mg/mlto 2.5 mg/ml, include propylene glycol in an amount of at least 70 vol %of the substantially non-aqueous solvent system, and further includeacetate buffer at a buffer strength of between 0.05 and 0.25M, whereinthe buffer has a pH between 2.7 and 3.3 (and most typically 3.0). Suchcompositions will exhibit equal or less than 10% degradation ofbortezomib at accelerated storage conditions after 3 months.

Regardless of the particular formulation, it is especially preferredthat the formulation is packaged in a container suitable for both singleand multi use. Thus, especially preferred containers include an ampoule,a vial, a pre-filled syringe, and intravenous bag. Especially preferredmulti-use containers will contain bortezomib in an amount suitable toallow at least two distinct uses, more typically at least five, and mosttypically at least ten distinct uses (each of which may or may notrequire the same quantity of formulation administered to the patient).Thus, particularly preferred containers will be configured as amulti-use container (e.g., contain a volume of the composition that issuitable for multiple and independent administrations), and especiallypreferred multi-use containers include vials with a rubber stopper thatcan be pierced with a needle of a syringe.

Thus, it should be appreciated that contemplated formulations willtypically allow storage of the bortezomib for at least 1 week afterfirst use, more typically at least 2-4 weeks after first use, and mosttypically at least 1-3 months (and even longer) after first use withoutsignificant degradation (i.e., less than 10% degradation, more typicallyless than 5% degradation) of the bortezomib under ambient conditions.Bortezomib may therefore be formulated for administration to human andvarious animals, and especially mammals. For example, formulations maybe in the form of a solution for injection (e.g., injectable multi dosesterile composition), in the form of a sterile powdered composition(e.g., lyophilized cake, powder, lyophilized powder), which may beadministered after dilution or reconstitution.

Therefore, the inventors also contemplate a method of suppressingformation of a plurality of degradation products of bortezomib insolution in which a single-phase liquid formulation is compounded from asubstantially non-aqueous solvent system suitable for injection, abuffer, and bortezomib, wherein the bortezomib is present in theformulation at a pharmaceutically effective concentration. Mostpreferably, the solvent system comprises as a main component propyleneglycol, and the solvent system, the buffer, and the pH are selected suchas to be effective to suppress formation of an amide degradationproduct, a first carbinolamide degradation product, and a secondcarbinolamide degradation product when the liquid formulation is storedunder storage conditions. With respect to the degradation products, thesolvent system, the buffer, and the pH, the same considerations as notedabove apply and are not reiterated here.

EXAMPLES

The following experiments are provided to exemplarily illustrate variousaspects of the inventive subject matter presented herein. However, itshould be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein.

Non-Aqueous Formulations (Set 1):

Five non-aqueous formulations were prepared with various ingredientsshown in Table 1. More particularly, a stock solution of D/L-Tocopherolwas made by dissolving 625 mg of D/L-Tocopherol in 25 ml of ethanol, anda stock solution of butylated hydroxytoluene (BHT) and butylatedhydroxyanisole (BHA) were prepared by dissolving 15 mg of each in 100 mlof ethanol, respectively. All five formulations were prepared bydissolving, 20 mg of bortezomib in 200 proof 10 ml ethanol and 100 μl ofDL Tocopherol ethanolic stock, 0.2 ml of BHT and BHA stock were addedaccordingly as per Table 1. Samples were then stored in an amber vialwith nitrogen head space and stored at various storage conditions asindicated in the tables. The pH for the Formulations I-V was 4.0

TABLE 1 Formulation Formulation Formulation Formulation FormulationIngredients I II III IV V Bortezomib 4.0 mg 4.0 mg 4.0 mg 4.0 mg  4.0 mgEthanol 2.0 ml 2.4 ml 2.2 ml 2.2 ml 10.0 ml Propylene 8.0 ml 7.6 ml 7.8ml 7.8 ml — Glycol Dl Tocopherol % 0.05 0.05 0.05 w/v Butylated 0.000030.00003 Hydroxy Toluene % w/v Butylated 0.00003 0.00003 Hydroxy Anisole% w/v

Stability results are shown in Tables 2-4, wherein Table 2 lists resultsfor the stability tests of bortezomib at 40° C. and 75% relativehumidity, Table 3 lists results for the stability tests of bortezomib at25° C. and 60% relative humidity, and Table 4 lists results for thestability tests of bortezomib at 4° C. Carbinolamide I is compound II ofScheme I; Carbinolamide II is compound III of Scheme I; Amide iscompound IV of Scheme I; Carboxylic acid is compound V of Scheme I

TABLE 2 Formulation I II III IV V Assay 1 Month 93 91 94 91 76 2 Month95 91 93 94 65 3 Month 93 91 92 92 51 Amide 1 Month 1.1 1.29 1.05 1.1312 2 Month 1.9 4.22 3.17 2.63 26 3 Month 3.2 3.94 3.86 3.36 41Carboxylic acid 1 Month 2.01 2.01 — 3.66 8.9 2 Month 0.99 1.40 1.38 1.305.4 3 Month 2.14 2.02 2.18 1.92 4.3 Carbinolamide I 1 Month 1.15 0.141.24 1.4 0.38 2 Month 1.18 2.21 1.88 1.52 0.07 3 Month 1.25 1.43 1.291.4 0.12 Carbinolamide II 1 Month — — — 0.08 — 2 Month 0.10 0.23 0.160.16 0.03 3 Month 0.15 0.19 0.18 0.17 0.03 Unknown (2.15 RRT) 1 Month —— — — — 2 Month 0.06 0.14 0.12 1.79 3 Month 0.17 0.19 0.19 2.40

TABLE 3 Formulation I II III IV V Assay 1 Month 94.39 94.41 95.2 95.290.6 2 Month 98.96 98.55 98.6 98.8 92.6 3 Month 98.75 98.07 98.24 98.4988.37 Amide 1 Month 0.1 0.13 0.08 0.11 1.38 2 Month 0.2 0.27 0.26 0.232.91 3 Month 0.27 0.38 0.35 0.33 5.44 Carboxylic acid 1 Month — — — —4.55 2 Month — — — — 3.62 3 Month — — — — 5.15 Carbinolamide I 1 Month0.73 0.91 0.8 0.94 — 2 Month 0.62 1.03 0.9 0.72 0.06 3 Month 0.77 1.041.21 1.04 0.07 Carbinolamide II 1 Month — — — — — 2 Month 0.06 0.08 0.070.07 — 3 Month 0.07 0.09 0.09 0.08 — Unknown (2.15 RRT) 1 Month — — — —— 2 Month — — — — 1.02 3 Month — — — — 0.77

TABLE 4 Formulation I II III IV V Assay 3 Month 99.47 99.31 99.38 99.4098.8 Amide (0.72 RRT) 3 Month 0.10 0.10 0.10 0.10 0.59 Carboxylic acid 3Month — — — — — Carbinolamide I 3 Month 0.36 0.48 0.40 0.39 0.40Carbinolamide II 3 Month 0.06 0.06 0.06 0.06 0.11 Unknown (2.15 RRT) 3Month 0.07

Non-Aqueous Formulations (Set 2):

Five additional formulations were prepared with various ingredients asshown in Table 5. The formulations were prepared as follows: Degas thewater for injection (WFI) to remove the dissolved oxygen in WFI andPropylene Glycol, refined Polyethylene Glycol 300 and Acetate buffer,weigh required amount of Bortezomib and add to the compounding vesseland dissolve in PG or PEG in the compounding vessel with stirring. Aftercomplete dissolution of the drug add remaining amount of vehicle such aspropylene glycol, polyethylene glycol, and buffer. In case of theformulation with N-acetyl cysteine, add and dissolve N-acetyl cysteinein buffer under nitrogen and add to the drug solution. For formulationsA-E the pH was 4.0

TABLE 5 Concentration Batch Ingredients (mg/mL) Quantity Formulation ABortezomib 1 25.0 mg Propylene Glycol qs 25 gm Formulation B Bortezomib1 25.0 mg Refined PEG qs 25 gm Formulation C Bortezomib 1 25.0 mgPropylene Glycol 45 22.5 gm Acetate Buffer 5 2.5 gm Formulation DBortezomib 1 25.0 mg Propylene Glycol 25 12.5 gm Acetate Buffer 25 12.5gm Formulation E Bortezomib 1 50.0 mg Propylene Glycol 25 25 gm AcetateBuffer 25 25 gm N-Acetyl Cysteine 5 0.250 gm

Stability results are shown in Tables 6-8, wherein Table 6 lists resultsfor the 2-week stability tests of bortezomib at indicated storageconditions, Table 7 lists results for the 6-week stability tests ofbortezomib at indicated storage conditions, and Table 8 lists resultsfor the 2-month stability tests of bortezomib at indicated storageconditions. ND=not detected using HPLC method as described above NA=notavailable; QL=Quantitation Limit; ND=Not Detected

TABLE 6 Formulation Initial 2 Week Storage Condition 25° C./60% RH 40°C./75% RH Formulation A Bortezomib (1 mg/ml) in 100% PG Assay % 100 10099.5 % Highest Impurity ND ND 0.13 Formulation D Bortezomib (1 mg/ml) in50% PG and 50% Acetate Buffer Assay % 99.65 99.86 99.26 % HighestImpurity ND 0.14 0.62 Formulation C Bortezomib (1 mg/ml) in 90% PG and10% Acetate Buffer Assay % 99.84 98.34 99.61 % Highest Impurity ND 0.18ND Formulation E Bortezomib (1 mg/ml) in 90% PG and 10% Acetate Bufferwith NAC Assay % 99.74 99.88 84.52 % Highest Impurity 0.16 0.12 7.11

TABLE 7 Formulation Initial 6 Week Storage Condition 0-4° C. 25° C./60%40° C./75% RH RH Formulation A Bortezomib (1 mg/ml) in 100% PG Assay %100 100 99.64 98.25 % Highest Impurity ND ND 0.14 0.65 Formulation DBortezomib (1 mg/ml) in 50% PG and 50% Acetate Buffer Assay % 99.6599.42 99.01 94.83 % Highest Impurity ND 0.08 0.42 1.93 Formulation CBortezomib (1 mg/ml) in 90% PG and 10% Acetate Buffer Assay % 99.8499.85 99.56 98.34 % Highest Impurity ND 0.16 0.51 Formulation EBortezomib (1 mg/ml) in 90% PG and 10% Acetate Buffer with NAC Assay %99.74 62.36 91.64 44.21 % Highest Impurity 0.16 1.16 0.28 27.42

TABLE 8 Formulation Initial 2 Month 3 Month Storage Condition 0-4° C.25° C./60% RH 40° C./75% RH 0-4° C. 25° C./60% 40° C./75% RH RHFormulation A Bortezomib (1 mg/ml) in 100% PG Assay % 100 99.79 99.3297.46 99.73 99.77 96.11 % Highest Impurity ND 0.07 0.23 0.98 <QL 0.111.7 Formulation D Bortezomib (1 mg/ml) in 50% PG and 50% Acetate BufferAssay % 99.65 98.66 97.67 88.66 99.55 98.69 86.06 % Highest Impurity ND0.11 0.72 3.29 0.07 0.98 4.73 Formulation C Bortezomib (1 mg/ml) in 90%PG and 10% Acetate Buffer Assay % 99.84 99.68 99.41 96.28 99.68 99.5894.65 % Highest Impurity 0.07 0.15 1.06 <QL <QL 1.74 Formulation EBortezomib (1 mg/ml) in 90% PG and 10% Acetate Buffer with NAC Assay %99.74 63 61 49.89 % Highest Impurity 0.16 26 20.47 31.7 NA NA

Formulation B with PEG was not included in the study due to insolubilityof the drug in PEG. As can be taken from the above results, stability ofbortezomib is enhanced in the presence of PG. Formulation with 10% ofaqueous buffer also showed a comparable stability to that of formulationwith PG alone. However, an increase in the buffer concentration hasrevealed an undesirable increase in degradation products. Notably, thepresence of a stabilizer/antioxidant like N-Acetyl Cysteine resulted ina significant degradation of the bortezomib.

Non-Aqueous Formulations (Set 3):

An additional six substantially non-aqueous formulations were preparedwith various ingredients as shown in Table 9 and Table 10. Theformulations were prepared as follows: Degas the buffer solutions andwater for injection to remove the dissolved oxygen, weigh requiredamount of Bortezomib and add to the compounding vessel and dissolve inPG in the compounding vessel with stirring. After complete dissolutionof the drug, add remaining amount of vehicle such as water for injectionand buffer. Samples were then filled in amber vials with nitrogen headspace and stored at 40° C./75% RH storage condition for duration of 3months.

TABLE 9 Formulation Formulation Formulation Formulation Ingredients I IIIII IV Bortezomib 10.0 mg 10.0 mg 10.0 mg 10.0 mg Propylene  9.0 mL  9.0mL  9.0 mL  9.0 mL Glycol Acetate Buffer —  1.0 mL — — (pH 4.0, 0.1M)Acetate Buffer — —  1.0 mL — (pH 4.0, 0.5M) Acetate Buffer — — —  1.0 mL(pH 4.0, 1M)) W.F.I  1.0 mL — — —

TABLE 10 Ingredients Formulation V Formulation VI Bortezomib 10.0 mg10.0 mg Acetate Buffer  5.0 mL  3.0 mL (pH 4.0, 0.1M) Propylene  5.0 mL 7.0 mL Glycol

Stability results are shown in Table 11, wherein Table 11 lists resultsfor the stability tests of bortezomib stored at 40° C. and 75% relativehumidity. The stability of these formulations were monitored todelineate the effect of buffer strength and the proportion of propyleneglycol on the product stability. ND=not detected using HPLC method asdescribed above; Carbinolamide I is compound II of Scheme I;Carbinolamide II is compound III of Scheme I; Amide is compound IV ofScheme I; Carboxylic acid is compound V of Scheme I.

TABLE 11 Formulation I II III IV V VI % Assay 1 Month 97.2 98.9 98.396.7 97.6 98.4 2 Month 88.5 95.7 93.0 91.0 78.7 92.8 3 Month 79.5 87.187.4 84.9 75.5 81.6 Amide 1 Month 0.43 0.11 0.13 0.16 0.18 0.08 2 Month0.60 0.14 0.20 0.24 0.28 0.12 3 Month 1.38 0.25 0.30 0.42 0.47 0.20Carboxylic Acid 1 Month 0.77 0.47 0.55 0.70 1.92 1.11 2 Month 1.00 0.670.81 1.00 2.50 1.56 3 Month 1.62 0.89 1.11 1.42 3.78 2.22 CarbinolamideI 1 Month 1.16 0.33 0.5 0.56 0.15 0.24 2 Month 1.38 0.46 0.56 0.75 0.150.29 3 Month 2.39 0.61 0.79 1.01 0.16 0.35 Carbinolamide II 1 Month 0.360.11 0.13 0.19 0.05 ND 2 Month 0.37 0.13 0.16 0.22 0.07 0.06 3 Month0.42 0.14 0.15 0.19 0.08 0.06

As can be taken from the above results, the stability of bortezomibseems to be compromised by increasing the buffer strength from 0.1M to 1M. The order of stabilization with respect to the buffer strength being0.1M>0.5M>1.0M. On a similar note, the formulations without any buffershowed decrease in the % assay and an increase in the related substancescompared to the one with 0.1 M acetate buffer. In formulations V and VI,where the composition of aqueous phase was fixed and the proportion ofPG was varied, the order of stabilization was 70% PG>50% PG.

Non-Aqueous Formulations (Set 4):

Several further compositions were prepared with various ingredients andvarious examples listed in Table 12. In this example, the possibleeffects of super-refined solvents on the stability of bortezomib wasinvestigated essentially as described above. Solutions were prepared asfollows: Degas the WFI to remove the dissolved oxygen in WFI andPropylene Glycol, Refined PG, refined Polyethylene Glycol 300 (PEG) andAcetate buffer, weigh required amount of Bortezomib and add to thecompounding vessel and dissolve in PG and PEG in the compounding vesselwith stirring to make 2 mg/ml solution. The stock solution was furtherdiluted to 1 mg/ml by adding remaining amount of vehicle such as PG, PEGand acetate buffer. pH of all the formulations in Table 12 was 4.0

TABLE 12 Initial Potency of the Formulation With PEG and PG Formulationwith Formulation with Super Refined Formulation with PG Super Refined PGPEG 1 mg/ml with 1 mg/ml with 1 mg/ml with 20% acetate 20% acetate 20%acetate 2 mg/ml 1 mg/ml Buffer 2 mg/ml 1 mg/ml Buffer 2 mg/ml 1 mg/mlBuffer % Assay 99.2 99.2 99.2 99.12 99.1 99.37 95.8 94.8 95.8 % 0.8 0.80.91 0.88 ND 0.63 4.2 5.17 4.19 Highest Impurity

Remarkably, the results indicate that there is no influence of the typeof PG used in the formulation. However, the inventors have observed asignificant degradation of bortezomib in the presence of super-refinedPEG. This indicates that bortezomib can be stabilized in presence ofpropylene glycol, but apparently cannot be stabilized in the presence ofa closely related alternative glycolic solvent, PEG under theexperimental parameters as shown.

Non-Aqueous Formulations (Set 5):

Five substantially non-aqueous formulations were prepared with variousingredients as shown in Table 13. In these example formulations, theproportion of propylene glycol was fixed at 90%, the variable being thecomposition of the aqueous phase. In these formulations, citrate buffer,pH 4.0 and phosphate buffer pH, 7.4, each at two levels of ionicstrength were used. Additionally, to measure the effect of ionicstrength on the product stability, NaCl was added to increase the ionicstrength to 0.5M. The formulations were prepared as follows: Degas thebuffer solutions to remove the dissolved oxygen, weigh required amountof Bortezomib and add to the compounding vessel and dissolve in PG inthe compounding vessel with stifling. After complete dissolution of thedrug add remaining amount of vehicle such as propylene glycol andbuffer. Samples were then filled in amber vials with nitrogen head spaceand stored at ‘super accelerated’ stability condition of 50° C. forduration of seven days.

TABLE 13 Formulation Formulation Formulation Formulation FormulationIngredients VII VIII IX X XI Bortezomib 10.0 mg 10.0 mg 10.0 mg 10.0 mg10.0 mg Propylene  9.0 mL  9.0 mL  9.0 mL  9.0 mL  9.0 mL Glycol Citratebuffer  1.0 mL — — — — (pH 4.0, 0.05M) Citrate buffer —  1.0 mL — — —(pH 4.0, 0.5M) Phosphate buffer — —  1.0 mL — — (pH 7.4, 0.05M)Phosphate buffer — — —  1.0 mL — (pH 7.4, 0.5M) Acetate Buffer — — — — 1.0 mL (pH 4.0, 0.1M) NaCl — — — — 0.29 g

Stability test results are shown in Table 14. Carbinolamide I iscompound II of Scheme I; Carbinolamide II is compound III of Scheme I;Amide is compound IV of Scheme I; Carboxylic Acid is compound V ofScheme I.

TABLE 14 Formulation VII VIII IX X* XI* % Assay 0 99.8 99.7 99.9 — — 3days 99.5 NT 98.8 — — 7 days 94.7 95.9 95.3 — — Amide 0 ND ND ND — — 3days 0.30 NT 0.18 — — 7 days 1.70 0.55 1.38 — — Carboxylic Acid 0 ND NDND — — 3 days 0.20 NT 0.16 — — 7 days 0.51 0.97 0.49 — — Carbinolamide I0 ND ND ND — — 3 days ND NT 0.06 — — 7 days 0.45 0.37 0.40 — —Carbinolamide II 0 ND ND ND — — 3 days ND NT 0.63 — — 7 days 1.33 0.401.22 — —

Addition of Propylene Glycol to the respective aqueous phases offormulations X and XI led to the precipitation of the buffer salts. Andhence, the stability analysis of these formulations was not feasible.Comparing the seven day accelerated stability results it was observedthat the formulations with phosphate buffer, pH 7.4 were the leaststable followed by the formulations with citrate buffer, pH 4.0(moderate stability).

Non-Aqueous Formulations (Set 6):

To further delineate the effect of pH on the stability of bortezomib,seven substantially non-aqueous formulations (F-XII to F-XVIII) wereprepared with various ingredients as shown in Table 15. In these exampleformulations the proportion of propylene glycol was fixed at 90%, whilevarying the pH of the aqueous phase from pH 2.2 to 5.0. The formulationswere prepared as follows: Degas the buffer solutions to remove thedissolved oxygen, weigh required amount of Bortezomib and add to thecompounding vessel and dissolve in PG in the compounding vessel withstirring. After complete dissolution of the drug add remaining amount ofvehicle such as propylene glycol and buffer. Samples were then filled inamber vials with nitrogen head space and stored at ‘super accelerated’stability condition of 50° C. for duration of 15 days.

TABLE 15 Ingredients F-XII F-XIII F-XIV F-XV F-XVI F-XVII F-XVIIIBortezomib 10.0 mg 10.0 mg 10.0 mg 10.0 mg 10.0 mg 10.0 mg 10.0 mgPropylene  9.0 mL  9.0 mL  9.0 mL  9.0 mL  9.0 mL  9.0 mL  9.0 mL GlycolAcetate Buffer —  1.0 mL — — — — — (pH 3.0, 0.1M) Acetate Buffer — — 1.0 mL — — — — (pH 5.0, 0.1M) Acetate Buffer — — —  1.0 mL — — — (pH4.0, 0.1M) Potassium — — — —  1.0 mL — — Chloride/Hcl Buffer (pH 2.2,0.1M) Potassium — — — —  1.0 mL — Hydrogen phthalate/HCl buffer (pH 2.2,0.1M) Citrate Buffer — — — — —  1.0 mL (pH 3.0, 0.1M) W.F.I, pH 4.0  1.0mL — — — — —

Stability results are shown in Table 16. Carbinolamide I is compound IIof Scheme I; Carbinolamide II is compound III of Scheme I; Amide iscompound IV of Scheme I; Carboxylic Acid is compound V of Scheme I.

TABLE 16 Formulation XII XIII XIV XV XVI XVII XVIII % Assay  0 101.2100.9 100.5 101.2 100.1 99.2 100.2  3 days 100.5 100.5 93.6 100.6 89.291.9 69.1  7 days 94.4 100.5 85.9 95.3 79.7 86.5 58.2 15 days NT 99.8 NTNT NT NT NT Amide  0 ND ND ND ND ND ND ND  3 days 0.08 ND 0.20 ND 0.950.59 1.74  7 days 0.13 ND 0.79 0.06 1.96 1.25 2.65 15 days — ND — — — —— Carboxylic Acid  0 ND ND ND ND ND ND ND  3 days 0.10 0.07 0.25 0.100.21 0.19 0.22  7 days 0.26 0.12 0.23 0.23 0.35 0.32 0.33 15 days — 0.42— — — — — Carbinolamide I  0 ND ND ND ND ND ND ND  3 days ND ND 0.72 ND0.05 ND 0.03  7 days 0.30 ND 0.14 0.16 0.02 ND 0.04 15 days — ND — — — —— Carbinolamide II  0 ND ND ND ND ND ND ND  3 days 0.08 ND 0.21 0.060.41 0.26 4.9  7 days 0.18 ND 0.12 0.10 0.60 0.38 3.58 15 days — ND — —— — —

The results of the 15 day ‘super-accelerated’ stability studies indicatethat in addition to the propylene glycol proportion, the pH of theaqueous phase has an effect on the stability of bortezomib.Specifically, for the formulations with aqueous phase pH 3.0, the %assay at the end of 15 days was at 99.8, the highest among all theformulations screened. In comparison, in the formulations with theaqueous phase pH of 5.0 and pH 2.2, Bortezomib exhibited rapiddegradation.

Non-Aqueous Formulations (Set 7):

A substantially non-aqueous formulations were prepared with variousingredients as shown in Table 17. The formulations were prepared asfollows: Degas the buffer solution and water for injection to remove thedissolved oxygen, weigh required amount of Bortezomib and add to thecompounding vessel and dissolve in PG in the compounding vessel withstifling to make Bortezomib solution, followed by addition ofappropriate amount of acetate buffer to make 2.5 mg/ml. The compositionswere exerted 4° C. and ambient temperatures to monitor the physicalstability. All the three compositions are physical stable, withFormulation XIX being the most preferred for administration ofBortezomib by subcutaneous route at a Bortezomib concentration of 2.5mg/ml.

TABLE 17 Formulation Formulation Formulation Ingredients XIX XX XXIBortezomib 25.0 mg 25.0 mg 25.0 mg Propylene  9.0 mL  7.0 mL  5.0 mLGlycol Acetate Buffer  1.0 mL  3.0 mL  5.0 mL (pH 3.0, 0.1M)

Therefore, particularly preferred formulations will include those thatare clear, colorless, sterile, self-preserved multi-dose, non-pyrogenicsolution, preferably in two concentrations, 1 mg and 2.5 bortezomib perml for intravenous (IV) and subcutaneous (SC) use respectively. Mostpreferably, such formulations will be provided in a 10-mL amber vialthat contains 1 mg/mL of Bortezomib and a 5-mL vial that contains 2.5mg/mL of bortezomib. Each vial of either size also contains 0.9 ml/1.0ml of Propylene Glycol USP and 0.1 ml/mL of pH 3.0, 0.1 molar aqueousacetate buffer as exemplarily shown in Table 18.

TABLE 18 Composition 1 Composition 2 Bortezomib   1 mg 2.5 mg PropyleneGlycol USP 0.9 mL 0.9 mL Aqueous Acetate Buffer, 0.1M, 0.1 mL 0.1 mL pH3.0

Non-Aqueous Formulations (Set 8):

Substantially non-aqueous formulations were prepared with variousingredients as shown in Table 19. The formulations were prepared asfollows: Degas the buffer solution and water for injection to remove thedissolved oxygen, weigh required amount of Bortezomib and add to thecompounding vessel and dissolve in PG in the compounding vessel withstifling to make Bortezomib solution, followed by addition ofappropriate amount of acetate buffer to make 1.0 mg/ml. The Bortezomibbulk solutions were filled into 10 mL amber type I vials with or withoutthe nitrogen headspace. The compositions were then subjected to 2-8° C.,ambient temperature (25° C./60% RH) and accelerated temperature (40°C./75% RH) to monitor the chemical stability, and results after 1 monthstorage are provided in Table 20.

TABLE 19 Formulation Formulation Formulation Ingredients XIX XX XXIBortezomib 100.0 mg 100.0 mg 100.0 mg Propylene  50.0 mL  70.0 mL  90.0mL Glycol Acetate Buffer  50.0 mL  30.0 mL  10.0 mL (pH 3.0, 0.1M)

TABLE 20 % Carboxylic Carbinol- Carbinol- Storage Headspace Sample NameAssay Amide Acid amide 1 amide 2 2-8° C. without N₂ Formulation XIX 98.7<QL <QL ND 0.08 Formulation XX 99.0 <QL ND ND ND Formulation XXI 100.2<QL ND ND <QL with N₂ Formulation XIX 99.6 <QL <QL ND <QL Formulation XX99.9 <QL ND ND ND Formulation XXI 97.5 <QL ND ND 0.05 25° C./60% RHwithout N₂ Formulation XIX 99.1 0.07 0.21 ND <QL Formulation XX 97.8 <QL0.08 ND ND Formulation XXI 97.0 <QL <QL ND 0.05 with N₂ Formulation XIX97.3 0.06 0.20 ND <QL Formulation XX 98.9 <QL 0.09 ND ND Formulation XXI98.8 <QL <QL ND ND 40° C./75% RH without N₂ Formulation XIX 97.0 0.491.34 <QL <QL Formulation XX 97.3 <QL 0.54 <QL ND Formulation XXI 98.5<QL 0.17 <QL <QL with N₂ Formulation XIX 97.6 0.32 1.16 <QL 0.18Formulation XX 99.5 <QL 0.55 <QL ND Formulation XXI 100.2 <QL 0.18 <QL<QL

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. An extended potency, ready to injectpharmaceutical parenteral composition in a vial, the compositioncomprising: a single-phase liquid formulation comprising a substantiallynon-aqueous solvent system suitable for injection, an aqueous buffer,and bortezomib, wherein the bortezomib is present in the formulation ata therapeutically effective concentration; wherein the single-phaseliquid formulation is formulated such that at least 95% of thebortezomib remain as active ingredient after storage at ambientconditions for at least three months; wherein the substantiallynon-aqueous solvent system comprises more than 50 vol % propyleneglycol; and wherein the liquid formulation and bortezomib are present inthe vial in a quantity suitable for at least two independent anddistinct administrations.
 2. The composition of claim 1 wherein thesubstantially non-aqueous solvent system consists essentially ofpropylene glycol.
 3. The composition of claim 1 wherein the formulationcomprises at least 75 vol % propylene glycol.
 4. The composition ofclaim 1 wherein the formulation comprises at least 90 vol % propyleneglycol.
 5. The composition of claim 1 wherein the buffer is an aqueousacetate buffer.
 6. The composition of claim 1 wherein the buffer has apH between 2.0 and 4.0.
 7. The composition of claim 1 wherein the bufferhas a pH that is equal or less than 4.0, and wherein the single-phaseliquid formulation has not undergone lyophilization before storage atambient conditions for at least three months.
 8. The composition ofclaim 1 wherein the bortezomib is present at a concentration of between1 mg/ml and 5 mg/ml.
 9. A method of stabilizing bortezomib in a liquidpharmaceutical composition that includes bortezomib in a therapeuticallyeffective amount, comprising: formulating a ready to inject single-phaseliquid formulation from a substantially non-aqueous solvent systemsuitable for injection, an aqueous buffer, and bortezomib; wherein thesolvent system comprises more than 50 vol % propylene glycol and whereinthe bortezomib is present in the formulation at a therapeuticallyeffective concentration; adjusting the pH of the buffer such that thesingle-phase liquid formulation has a pH that is equal or less than 4.0;and placing the single-phase liquid formulation in a vial, optionally ina quantity suitable for at least two independent and distinctadministrations.
 10. The method of claim 9 wherein the single-phaseliquid formulation is formulated such that at least 98% of thebortezomib remain as active ingredient after storage at ambientconditions for at least three months.
 11. The method of claim 10 whereinthe single-phase liquid formulation comprises ethanol and propyleneglycol, or exclusively propylene glycol.
 12. The method of claim 9wherein the single-phase liquid formulation includes the aqueous bufferand is formulated such that at least 98% of the bortezomib remain asactive ingredient after storage at ambient conditions for at least threemonths.
 13. The method of claim 12 wherein the single-phase liquidformulation comprises at least 75 vol % propylene glycol.
 14. The methodof claim 13 wherein the aqueous buffer is acetate buffer.
 15. The methodof claim 12 wherein the pH of the buffer is adjusted such that thesingle-phase liquid formulation has a pH that is between 2.7. and 3.3.16. A method of maintaining potency of a liquid pharmaceuticalcomposition that contains bortezomib by reducing degradation ofbortezomib in a liquid formulation, the method comprising: preparing asingle-phase ready to inject liquid formulation from a substantiallynon-aqueous solvent system suitable for injection, and bortezomib;wherein the substantially non-aqueous solvent system comprises more than50 vol % propylene glycol; including an aqueous buffer into theformulation and adjusting the pH of the buffer to a value between 2.0and 4.0 to thereby reduce formation of at least one degradation productas compared to the formulation without buffer; and wherein the at leastone degradation product is selected form the group consisting of anamide degradation product, a carboxylic acid degradation product, afirst carbinolamide degradation product, and a second carbinolamidedegradation product; and placing the single-phase liquid formulation ina multi-use vial, optionally in a quantity suitable for at least twoindependent and distinct administrations.
 17. The method of claim 16wherein the aqueous buffer is selected from the group consisting of anacetate buffer, a citrate buffer, and a potassium hydrogen phthalate/HClbuffer.
 18. The method of claim 16 wherein the pH of the buffer isadjusted to a value between 2.7 and 3.3 to thereby reduce formation ofthe amide degradation product, the carboxylic acid degradation product,the first carbinolamide degradation product, and the secondcarbinolamide degradation product.
 19. The method of claim 18 whereinthe buffer is an acetate buffer.
 20. The method of claim 16 wherein thebortezomib is present in the liquid pharmaceutical composition at aconcentration of 1 mg/ml and 5 mg/ml.